Forskolin (Fsk), a diterpene natural product, interacts directly with adenylyl cyclase in diverse tissues to cause an increase in intracellular cyclic AMP. There are at least nine distinct mammalian adenylyl cyclases, each of which consist of two intensely hydrophobic domains (M1 and M2) and two cytoplasmic domains (C1 and C2). Dr. W.J. Tang has made available to us purified C1a and C2a domains of type I and type II adenylyl cyclases, respectively, which result in Fsk-activated enzyme activity when mixed together in vitro. We have been examining whether an alkylating (isothionyl) derivative of Fsk, 6-NCS-Fsk, may be used to identify the reactive nucleophilic group at the Fsk interaction site(s) of type I and type II adenylyl cyclase, as well as the mixed C1a/C2a domains. Although 6-NCS-Fsk inhibits high affinity Fsk binding to type I adenylyl cyclase, it does not stimulate activation of the type I enzyme. However, pretreatment with 6-NCS-Fsk causes a concentration-dependent irreversible loss of Fsk-stimulated activation of type I adenylyl cyclase. This result suggests that covalent modification of a nucleophilic group at the Fsk site of action by 6-NCS-Fsk prevents activation of the adenylyl cyclase by the Fsk moiety of 6-NCS-Fsk as well as Fsk itself. In contrast, 6-NCS-Fsk stimulates type II adenylyl cyclase activity as well as Fsk does, suggesting the absence of a nucleophilic group at the Fsk activation site of the type II enzyme. 6-NCS-Fsk does not stimulate activation of the mixed C1a/C2a domains, indicating that the noncovalent chimera is type I-like in its sensitivity. This result suggests that 6-NCS-Fsk is covalently modifying a nucleophilic group (most likely a histidine) in the C1a domain of type I adenylyl cyclase. We have made progress in attempting to identify the reactive amino acid at theFsk interaction site of adenylyl cyclase. We have established the conditions for generating proteolytic (endo-lys C) fragments of recombinant type I adenylyl cyclase. We are in the process of developing a protocol using anti-Fsk antibodies to immunoprecipite the peptide tagged with 6-NCS-Fsk. We are concentrating on isolating the chemically modified peptide from the mixed soluble C1a/C2a domains because they have been purified. However, it will be important to determine if the same nucleophilic residue is being covalently modified by 6-NCS-Fsk in intact type I adenylyl cyclase.